1. Field of the Invention
The present invention relates, in general, to a kinase which in its activated state is capable of site-specific phosphorylation of I.kappa.B.alpha., I.kappa.B.alpha. kinase. In particular, the present invention relates to the purified kinase, purified polypeptide subunits of the kinase, nucleic acid molecules coding for the purified polypeptide subunits; recombinant nucleic acid molecules; cells containing the recombinant nucleic acid molecules; antibodies having binding affinity specifically to the kinase or its polypeptide subunits; hybridomas containing the antibodies; nucleic acid probes for the detection of the nucleic acid encoding the kinase; a method of detecting nucleic acids encoding the kinase or polypeptides of the kinase in a sample; and kits containing nucleic acid probes or antibodies. This invention further relates to bioassays using the nucleic acid sequence, protein or antibodies of this invention to diagnose, assess, or prognose a mammal afflicted with an undesired activation of NF-.kappa.B. This invention also relates to ligands, agonists, and antagonists of the kinase, and diagnostic and therapeutic uses thereof. This invention also relates to bioassays using the kinase or polypeptides of the kinase of this invention to identify ligands, agonists, and antagonists. More specifically, this invention relates to selective inhibitors of the kinase and to structure-based design of ligands, agonists, and antagonists of the kinase. This invention further relates to gene therapy using the nucleic acids of the invention.
2. Related Art
Regulation of the immune and inflammatory responses requires the activation of specific sets of genes by a variety of extracellular signals. These signals include mitogens (e.g., LPS and PMA), cytokines (e.g., TNF-.alpha. and IL-1.beta.), viral proteins (e.g., HTLV-1 Tax), antigens, phosphatase inhibitors (e.g., okadaic acid and calyculin A), and UV light. The rel/NF-.kappa.B family of transcriptional activator proteins plays an essential role in the signal transduction pathways that link these signals to gene activation (reviewed by Siebenlist, U. et al., Annu. Rev. Cell. Biol. 10:405-455 (1994); Baerle & Henkel, Annu. Rev. Immunol. 12:141-179 (1994); Thanos & Maniatis, Cell 80:529-532 (1995); Finco & Baldwin, J. Biol. Chem. 24:17676-17679 (1993); Verma, I. M. et al., Genes & Dev. 9:2723-2735 (1995)). NF-.kappa.B (p50/RelA(p65)), and other heterodimeric rel family proteins are sequestered in the cytoplasm through their association with I.kappa.B.alpha. or I.kappa.B.beta., members of the I.kappa.B family of inhibitor proteins. In the case of I.kappa.B.alpha., and most likely I.kappa.B.beta., stimulation of cells leads to rapid phosphorylation and degradation of the inhibitor. Consequently NF-.kappa.B is released and translocates into the nucleus where it activates the expression of target genes. Phosphorylation of I.kappa.B.alpha. per se is not sufficient to dissociate NF-.kappa.B from the latent complex (Palombella, V. J., Cell. 78:773-785 (1994); Traenckner, E. B.-M. et al., EMBO J. 13:5433-56441 (1994); Finco, T. S. et al., Proc. Natl. Acad. Sci. USA 91:11884-11888 (1994); Miyamoto, S. et al., Proc. Natl. Acad. Sci. USA 91:12740-12744 (1994); Lin, Y.-C. et al., Proc. Natl. Acad. Sci. USA 92:552-556 (1995); Alkalay, I. et al., Mol. Cell. Biol. 15:1294-1304 (1995); DiDonato, J. A. et al., Mol. Cell. Biol. 15:1302-1311 (1995)). Rather, phosphorylation triggers the degradation of I.kappa.B.alpha. (Brown, K. et al., Science 267:1485-1491 (1995); Brockman, J. A. et al., Mol. Cell. Biol. 15:2809-2818 (1995); Traenckner, E. B.-M. et al, EMBO J. 14:2876-2883 (1995); Whiteside, S. T. et al., Mol. Cell. Biol. 15:5339-5345 (1995)).
Recently, it has been shown that signal-induced degradation of I.kappa.B.alpha. is mediated by the ubiquitin-proteasome pathway (Chen, Z. J. et al., Genes & Dev. 9:1586-1597 (1995); Scherer, D. C. et al., Proc. Natl. Acad. Sci. USA 92:11259-11263 (1995); Alkalay, I. et al., Proc. Natl. Acad. Sci. USA. 92:10599-10603 (1995)). In this pathway, a protein targeted for degradation is first modified by covalent attachment of ubiquitin, a highly conserved polypeptide of 76 amino acids (reviewed by Hershko & Ciechanover, Annu. Rev. Biochem. 61:761-807 (1992); Ciechanover, A., Cell 79:13-21 (1994)). Ubiquitination is a three-step process: First, ubiquitin is activated by a ubiquitin activating enzyme (E1); the activated ubiquitin is then transferred to a ubiquitin carrier protein (E2, also referred to as ubiquitin conjugating enzyme or UBC); finally, ubiquitin is conjugated to a protein substrate by forming an isopeptide bond between the carboxyl terminal glycine residue of ubiquitin and the .epsilon.-amino group of one or more lysine residues of the protein substrate. This conjugation step often requires a ubiquitin protein ligase (E3). Multiple molecules of ubiquitin can be ligated to a protein substrate to form multi-ubiquitin chains, which are then recognized by a large, ATP-dependent protease (MW .about.2000 kDa) called the 26S proteasome. The 26S proteasome is composed of a 20S catalytic core, and a 19S regulatory complex (reviewed by Goldberg, A. L., Science 268:522-523 (1995)).
Multiple E2s and E3s function together to mediate the ubiquitination of a variety of cellular proteins. For example, there are at least a dozen E2s in yeast that display distinct substrate specificities and carry out distinct cellular functions. The closely related E2 proteins UBC4 and UBC5 are involved in the turnover of many short-lived and abnormal proteins, and they play an essential role in the stress response (Seufert & Jentsch, EMBO J. 9:543-550 (1990)). Homologs of UBC4/UBC5 mediate the ubiquitination of the P53 protein in conjunction with the HPV-16 E6-E6AP complex, which functions as an E3 (Schaffner, M. et al., Cell. 75:495-505 (1993)). These E2s have also been implicated in the ubiquitination of the MAT.alpha.2 Processor (Chen, P. et al., Cell 74:357-369 (1993)), cyclin B (King, R. W. et al., Cell 81:279-288 (1995)), and the NF-.kappa.B precursor protein P105 (Orian, A. et al., J. Biol. Chem. 270:21707-21714 (1995)). The involvement of UBC4/UBC5 in the ubiquitination of such diverse substrates indicates that these E2s alone cannot confer substrate specificity. However, they may act together with specific E3s to recognize specific substrates. Although relatively few E3s have been identified thus far, the existence of a large family of these proteins is likely (Huibregtse, J. M. et al., Proc. Natl. Acad Sci. USA 92:2563-2567 (1995)).
Ubiquitination of I.kappa.B.alpha. is regulated by signal-induced phosphorylation at two specific residues, serines 32 and 36 (Chen, Z. J. et al., Genes & Dev. 9:1586-1597 (1995)). Single amino acid substitutions of one or both of these residues abolish the signal-induced phosphorylation and degradation of I.kappa.B.alpha. (Brown, K. et al., Science 267:1485-1491 (1995); Brockman, J. A. et al., Mol. Cell. Biol. 15:2809-2818 (1995); Traenckner, E. B.-M. et al., EMBO J. 14:2876-2883 (1995); Whiteside, S. T. et al., Mol. Cell. Biol. 15:5339-5345 (1995)). The same mutations also abolish the okadaic acid-induced phosphorylation and ubiquitination of I.kappa.B.alpha., in vitro (Chen, Z. J. et al., Genes & Dev. 9:1586-1597 (1995)). Relatively little is known about the signal transduction pathways and the kinase(s) responsible for the site-specific phosphorylation of I.kappa.B.alpha.. Mutants of I.kappa.B.alpha. lacking serines 32 and 36 are resistant to induced phosphorylation by a variety of stimuli, suggesting that different signal transduction pathways converge on a specific kinase or kinases. However, despite considerable effort, the identification of this I.kappa.B.alpha. kinase has remained elusive (Verma, I. M. et al., Genes & Dev. 9:2723-2735 (1995)).
Several serine/threonine kinases, including protein kinase C (PKC), heme-regulated eIF-2.alpha. kinase (HRI), protein kinase A (Ghosh & Baltimore, Nature 344:678-682 (1990)), casein kinase II (Barroga, C. F. et al., Proc. Natl. Acad Sci. USA 92:7637-7641 (1995)) and a recently described 42 kDa kinase (Kano, K. et al., J Biol. Chem. 270:27914-27919 (1995)), have been shown to phosphorylate I.kappa.B.alpha. in vitro. However, none of these kinases have been shown to phosphorylate I.kappa.B.alpha. at serines 32 and 36. Various kinases have also been implicated in the regulation of NF-.kappa.B in vivo, such as .zeta.PKC (Dominguez, I. et al., Mol. Cell. Biol 13:1290-1295 (1993); Diaz-Meco, M. T. et al., EMBO J. 13:2842-2848 (1994)), ceramide-dependent protein kinase (Schutze, S. et al., Cell 71:765-776 (1992)), tyrosine kinases (Devary, Y. et al., Science 261:1442-1445 (1993)), Raf (Finco & Baldwin, J. Biol. Chem. 24:17676-17679 (1993); Li & Sedivy, Proc. Natl. Acad. Sci. USA 90:9247-9251 (1993)), and the Drosophila pelle kinase, which is required for the inactivation of cactus, a Drosophila I.kappa.B homolog (Wassermann, S. A., Mol. Biol. Cell 4:767-771 (1993)). These kinases may function at various steps in the signal transduction pathway upstream of I.kappa.B.alpha. phosphorylation, but none have been shown to directly phosphorylate I.kappa.B.alpha. at relevant sites.
Although the diverse nature of NF-.kappa.B stimuli suggests that the initial steps in the signal transduction pathways are distinct, these pathways appear to converge on the generation of reactive oxygen intermediates (ROIs, such as H.sub.2 O.sub.2), which are thought to function as common second messenger-like molecules in the activation of NF-.kappa.B (Schreck, R. et al., EMBO J. 10:2247-2258 (1991)). At present the mechanistic link between ROIs and I.kappa.B.alpha. phosphorylation is not understood.
The establishment of an in vitro system for signal-induced phosphorylation and ubiquitination of I.kappa.B.alpha. was previously reported (Chen, Z. J. et al., Genes & Dev. 9:1586-1597 (1995)). The present invention involves fractionating HeLa cell cytoplasmic extracts and assaying for specific phosphorylation and ubiquitination of I.kappa.B.alpha.. The present invention provides a high molecular weight kinase complex that, in its activated state, specifically phosphorylates I.kappa.B.alpha. at serines 32 and 36. Surprisingly, UBC4/UBC5, ubiquitin, and E1 are not only involved in the ubiquitination of I.kappa.B.alpha., but may also be required for the phosphorylation of I.kappa.B.alpha.. Additional experiments reveal that this I.kappa.B.alpha. kinase can be activated by a prior ubiquitination event. In this case, ubiquitination serves a regulatory function without involving proteolysis. Additionally, this I.kappa.B.alpha. may be activated by MEKK1.